Stirred tank reactor systems and methods of use

ABSTRACT

A reactor system includes a support housing having an interior surface bounding a chamber, the chamber having a vertically extending central longitudinal axis. A flexible bag is disposed within the chamber of the support housing and has an interior surface bounding a compartment. A mixing element is disposed within the compartment of the flexible bag. A drive element, such as a drive shaft, is secured the mixing element, wherein the mixing element is laterally offset from and/or is angled relative to the vertically extending central longitudinal axis of the support housing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 15/376,362,filed Dec. 12, 2016, U.S. Pat. No. 10,640,741, which is a continuationof application Ser. No. 14/109,684, filed Dec. 17, 2013, U.S. Pat. No.9,540,060, which is a continuation of application Ser. No. 13/443,391,filed Apr. 10, 2012, U.S. Pat. No. 8,623,640, which is a divisional ofapplication Ser. No. 13/014,575, filed Jan. 26, 2011, U.S. Pat. No.8,187,867, which is a divisional of application Ser. No. 12/116,050,filed May 6, 2008, U.S. Pat. No. 7,901,934, which is a divisional ofapplication Ser. No. 11/112,834, filed Apr. 22, 2005, U.S. Pat. No.7,384,783, which claims the benefit of Provisional Application Ser. No.60/565,908, filed Apr. 27, 2004, which applications are incorporatedherein in their entirety by specific reference.

BACKGROUND OF THE INVENTION 1. The Field of the Invention

The present invention relates to a stirred-tank reactor system andmethods of use. The present invention further encompasses the use of thestirred-tank reactor system as a disposable bioreactor and in kits withdisposable elements.

2. The Relevant Technology

Bioreactors or fermenters include containers used for fermentation,enzymatic reactions, cell culture, biologicals, chemicals,biopharmaceuticals, tissue engineering, microorganisms, plantmetabolites, food production and the like. Bioreactors vary in size frombenchtop fermenters to stand-alone units of various sizes. The stringentasepsis requirements for sterile production in some bioreactors canrequire elaborate systems to achieve the desired product volumes.Consequently, the production of products in aseptic bioreactors can becostly which provides the motivation for pursuing improved systems.

Conventional bioreactors perfuse nutrient media through a single type ofhollow fiber. The various disadvantages of such bioreactors may includeheterogeneous cell mass, difficult procurement of representative cellgrowth samples, poor performance due to inefficient oxygenation and aninability to control oxygen levels, and problems with contamination ofcell cultures. Moreover, micro-environmental factors such as pH may notbe effectively controlled and a mixed culture or co-culture of cells maynot be possible. Some known bioreactors include a reaction container,through which a central strand of porous hollow fibers extends, throughwhich a nutrient solution is pumped. This central strand of hollowfibers is concentrically surrounded by a plurality of strands of hollowfibers, through which a gaseous medium is conveyed. The hollow fibers ofthese strands are also constituted in such a manner that the gaseousmedium—for example oxygen or carbon dioxide—can at least partly emergefrom these strands or enter into these strands respectively. This typeof bioreactor can achieve enhanced nutrient media oxygenation ascompared to other known devices. However, occasional contamination ofcell cultures and an inability to control pH levels effectively maycontinue to present difficulties.

The expense of producing cells, biopharmaceuticals, biologicals and thelike in aseptic bioreactors is often exacerbated by the requiredcleaning, sterilization and validation of the standard bioreactors(i.e., stainless steel or glass reactors). Attempts have been made tosolve this problem with the development of pre-sterilized disposablebioreactor systems that need not be cleaned, sterilized or validated byend users. The use of such disposable bioreactor systems could providesignificant savings. Furthermore, plastics are lightweight, easy totransport, and require less room than stainless steel or glass reactors.Some have reported the use of disposable elements in bioreactors thatinclude a reactor chamber with a support housing. The interior chamberof the support housing is lined with a disposable liner and sealed witha head plate attached to the liner to form a sealed chamber. As theliner is open at the top, it is typically used in a vertically orientedbioreactor to prevent the contamination of the head plate. Although thissystem provides a disposable liner, the head plate and the interiorchamber may still require cleaning and sterilization.

Others have attempted to develop flexible, disposable plastic vesselsthat do not require cleaning or sterilization and require only minimalvalidation efforts. Such approaches can include a flexible, disposable,and gas permeable cell culture chamber that is horizontally rotated. Thecell culture chamber is made of two sheets of plastic fused together. Inaddition, the culture chamber is made of gas permeable material and ismounted on a horizontally rotating disk drive that supports the flexibleculture chamber without blocking airflow over the membrane surfaces. Thechamber is placed in an incubator and oxygen transfer is controlled bycontrolling the gas pressure in the incubator according to thepermeability coefficient of the bag. The rotation of the bag assists inmixing the contents of the bag. However, the cell culture chamber willoften be limited to use within a controlled gas environment.Particularly, the cell culture chamber may have no support apparatus andmay be limited to small volumes. Furthermore, the chamber may notprovide an inlet and an outlet for media to be constantly pumped intoand out of the chamber during rotation.

Some companies have developed a range of pre-sterile, disposablebioreactors that do not require cleaning or sterilizing. Such reactorsare made of sheets of flexible, gas impermeable material to form a bag.The bag is partially filled with media and then inflated with air thatcontinually passes through the bag's headspace. The media is mixed andaerated by rocking the bags to increase the air-liquid interface.However, since there is typically no solid housing that supports thebags, the bags may become cumbersome and difficult to handle as theyincrease in size. Furthermore, the wave action within the rocking bagcan create damaging turbulent forces. Certain cell cultures,particularly human cell cultures, may benefit from more gentleconditions.

Thus, there is a continuing need to develop flexible, pre-sterilized,disposable bioreactors that are easy to handle and require littletraining to operate, yet provide the necessary gas transfer and nutrientmixing required for successful cell and tissue cultures. Such disposablebioreactors would be equally useful for the production of chemicals,biopharmaceuticals, biologicals, cells, microorganisms, plantmetabolites, foods and the like.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a stirred-tank reactorsystem with disposable elements, such as a flexible plastic bag with anattached bearing, shaft, and impeller assembly. The instant inventionfurther relates to the use of this novel stirred-tank reactor system asa disposable bioreactor and in kits with disposable elements. Theadvantages of the present invention are numerous. Particularly, thestirred-tank reactor system may be pre-sterilized and does not require asteam-in-place (SIP) or clean-in-place (CIP) environment for changingfrom batch to batch or product to product in a culture or productionsystem. As such, the system may require less regulatory control byassuring zero batch-to-batch contamination and can, thus, be operated ata considerable cost-advantage and with minimal or no preparation priorto use. In addition, the system can be a true stirred-tank reactorsystem unlike other disposable reactor systems. This provides the addedadvantage that the instant invention can offer a hydrodynamicenvironment that can be scaled to various sizes similar to conventionalnon-disposable reactor systems. As the system typically does not requirecleaning or sterilizing, it combines a flexible, easy-to-use, truestirred-tank reactor environment with zero cross-contamination duringthe cell culture or production process.

One aspect of the present invention provides a stirred-tank reactorsystem, comprising a flexible bag with at least one opening, wherein thebag functions as a sterile container for a fluidic medium; a shaftsituated within the bag; an impeller attachable to the shaft, whereinthe impeller is used to agitate the fluidic medium to provide ahydrodynamic environment; and a bearing attached to the shaft and to theopening of the bag. The bag may be affixed to the shaft and the bearingthrough at least one seal or O-ring such that the inside of the bagremains sterile. The seals or O-rings can be affixed to the bag. Thesystem may be disposable and pre-sterilized. The bag may further includea pH sensor and a dissolved-oxygen sensor, wherein the sensors areincorporated into the bag. In addition, the system may include at leastone internal pouch sealed to the bag, wherein the pouch has one end thatcan be opened to the outside of the bag such that a probe (i.e., atemperature probe, a pH probe, a dissolved gas sensor, an oxygen sensor,a carbon dioxide (CO₂) sensor, a cell mass sensor, a nutrient sensor, anosmometer, and the like) can be inserted into the reactor. The systemmay also include at least one port in the bag allowing for theconnection of a device such as a tube, a filter, a sampler, a probe, ora connection device to the port. A port allows for sampling; gas flow inand out of the bag; liquid or media flow in and out of the bag;inoculation; titration; adding of chemostat reagents; sparging; and thelike.

Another aspect of the present invention provides a stirred-tank reactorsystem, comprising a flexible bag with at least one opening, wherein thebag functions as a sterile container for a fluidic medium; a shaftsituated within the bag; an impeller attachable to the shaft, whereinthe impeller is used to agitate the fluidic medium to provide ahydrodynamic environment; and a bearing attached to the shaft and to theopening of the bag. The system may further include a housing, such as areactor housing, on the outside of the bag, wherein the housing includesat least one support that holds the bearing and a motor, and wherein thebag is contained within the housing. The housing may further include aplurality of baffles such that the bag folds around the baffles.Optionally, the system further encompasses a heater (e.g., a heatingpad, a steam jacket, a circulating fluid or water heater, etc.) that canbe located between the bag and the housing. Alternatively, the heatermay be incorporated into the housing (e.g., a permanent reactor housingwith incorporated heating system).

In another aspect of the invention, the stirred-tank reactor systemincludes a permanent housing with a product loop with flow past a pHsensor and a dissolved-oxygen sensor, wherein the sensors areincorporated into the housing. The permanent housing includes, but isnot limited to, a metal barrel, a plastic barrel, a wood barrel, a glassbarrel, and the like.

The invention also contemplates a method for preparing a stirred-tankreactor system, comprising providing a flexible bag with at least oneopening, wherein the bag functions as a sterile container for a fluidicmedium; inserting a shaft with an impeller attachable to the shaft intothe bag, wherein the impeller is used to agitate the fluidic medium toprovide a hydrodynamic environment; attaching a bearing to the shaft andto the opening of the bag; and sealing the bag to the shaft and thebearing such that the inside of the bag remains sterile. Thestirred-tank reactor system prepared by this method includes at leastone disposable element including, but not limited to, the bag, theshaft, the impeller, and the bearing.

The invention further encompasses a kit comprising a stirred-tankreactor system and instructions for use. The kit includes a disposablestirred-tank reactor system. The kit may also include a stirred-tankreactor system with at least one disposable element such as the bag, theshaft, the impeller, or the bearing. The bag may be affixed to the shaftand the bearing through at least one seal or O-ring such that the insideof the bag remains sterile. Furthermore, the bag may include a pH sensorand a dissolved-oxygen sensor, wherein the sensors are incorporated intothe bag. The kit may also include at least one internal pouch sealed tothe bag, wherein the pouch includes one end that can be opened to theoutside of the bag such that a probe can be inserted into the reactor.In addition, the system may include at least one port in the bagallowing for the connection of a device to the port, wherein the deviceincludes, but is not limited to, a tube, a filter, a sampler, and thelike.

Another aspect of the invention provides a bag for use in a stirred-tankreactor system. The bag may be a disposable, flexible, plastic bag. Thebag may also include at least one disposable element including, but notlimited to, a seal, an O-ring, a port, a pouch, a tube, a filter, asampler, a probe, a sensor, a connection device, or the like.

In one aspect, the present invention provides a reactor system thatincludes a container and a rotational assembly. The rotational assemblycan be in sealed cooperation with an opening of a container. Therotational assembly can include a rotatable hub adapted to receive andreleasably couple with a drive shaft, such that when the drive shaft isoperatively coupled with the rotatable hub, rotation of the drive shaftfacilitates a corresponding rotation of the rotatable hub. In a relatedaspect, the system can further include an impeller coupled with therotatable hub, such that the impeller is disposed within the containerand adapted to couple with a distal end of the drive shaft. In otheraspects, the rotational assembly can include a casing, whereby therotational assembly is in sealed cooperation with the opening of thecontainer via the casing. Similarly, the system can include a driveshaft, wherein the rotatable hub and the drive shaft are disposed torotate relative to the casing. In still a related aspect, the rotationalassembly can include a bearing assembly disposed between the casing andthe rotatable hub. The rotational assembly may further include a sealingarrangement disposed circumferentially to the rotatable hub, between therotatable hub and the casing. Relatedly, the bearing assembly caninclude a plurality of race bearings, and the sealing arrangement caninclude a rotating disk coupled with the rotatable hub, a wear platecoupled with the casing, and a dynamic seal disposed between therotating disk and the wear plate. In other aspects, a seal can includetwo or more seal subunits disposed in co-planar arrangement. Relatedly,a bearing assembly can include a journal bearing, and the sealingarrangement can include a wear plate coupled with the rotatable hub, anda dynamic seal disposed between the casing and the wear plate. In asimilar aspect, the impeller can include a spline adapted to couple withthe drive shaft. Often, the container can comprise a flexible bag. Inanother aspect, the rotatable hub can be coupled with the impeller via aflexible tube.

In one aspect, the present invention provides a reactor system thatincludes a container and a sparger assembly. The sparger assembly can bedisposed within the container, and can include a flexible sheet ofpermeable material and a sparger conduit. In a related aspect, the sheetof permeable material can include a vapor-permeable and water-resistantmaterial. In some aspects, the sheet of permeable material can include ahigh density polyethylene fiber. In related aspects, the spargerassembly can be in fluid communication with a port of the container.Similarly, the reactor system may include a rotational assembly insealed cooperation with an opening of the container, and an impellerdisposed within the container and coupled with the rotational assembly.The sparger body may be anchored to an interior surface of thecontainer, and in some cases, the sparger body of the sparger assemblycan be in a substantially spherical shape.

In another aspect, the present invention provides a bioreactor systemthat includes a frame support coupled with a drive motor; a flexible bagdisposed within a housing of the frame support. The flexible bag caninclude one or more ports for introducing a cell culture and a mediuminto the flexible bag; a rotational assembly coupled with a bracket ofthe frame support and in sealed cooperation with an opening of theflexible bag. The rotational assembly can include a hub adapted to houseand couple with a drive shaft of the drive motor. The system can alsoinclude an impeller coupled with the hub for agitating the cell cultureand medium. The impeller can be disposed within the flexible bag andadapted to couple with the drive shaft. In one aspect, the bioreactorsystem can include a probe assembly. The probe assembly can include aport coupled with the flexible bag, a PALL connector coupled with theport, a sleeve coupled with the PALL connector, a coupler coupled withthe sleeve, and a probe configured to be coupled with the coupler andinserted through the sleeve, PALL connector, and port, and partiallyinto the flexible bag.

In one aspect, the present invention provides a method for manufacturinga reactor system. The method can include coupling a container with arotational assembly. The rotational assembly can be in sealedcooperation with an opening of the container. The rotational assemblycan include a hub adapted to house and couple with a drive shaft. Themethod may also include coupling an impeller with the hub, where theimpeller is disposed within the container. The method may furtherinclude sterilizing the reactor system. In a related aspect, thesterilizing step can include treating the system with gamma radiation.

In another aspect, the present invention provides a method for preparinga reactor system. The method can include coupling a casing of arotational assembly of the reactor system to a frame bracket. The methodcan also include placing a container of the reactor system at leastpartially within a frame housing, and inserting a drive shaft into a hubof the rotational assembly. The hub can be disposed within the casing ofthe rotational assembly between a bearing and the casing. The method canfurther include coupling a distal end of the drive shaft to an impeller.The impeller can be disposed within the container and coupled with thehub. The method can also include introducing a reaction component intothe container via a port.

In one embodiment, the present invention provides a reactor system kit.The kit can have a reactor system that includes a container. The reactorsystem can also include a rotational assembly in sealed cooperation withan opening of the container. The rotational assembly can include a hubadapted to house and couple with a drive shaft, and an impeller coupledwith the hub. The impeller can be disposed within the container andadapted to couple with the drive shaft. The kit also includesinstructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is best understood when read in conjunction withthe accompanying figures which serve to illustrate the preferredembodiments. It is understood, however, that the invention is notlimited to the specific embodiments disclosed in the figures.

FIG. 1 depicts a longitudinal cross-section of one embodiment of thestirred-tank reactor system, wherein the stirred-tank reactor system isplaced into a permanent housing.

FIG. 2 depicts one embodiment of a probe connection in order toillustrate that a probe can be attached to the stirred-tank reactorsystem via a sterile or aseptic connection.

FIGS. 3A and 3B illustrate cross-section views of a reactor systemaccording to one embodiment of the present invention.

FIG. 4A illustrates a cross-section view of a rotational assemblyaccording to one embodiment of the present invention.

FIG. 4B illustrates a cross-section view of a rotational assemblyaccording to one embodiment of the present invention.

FIG. 5 illustrates a cross-section view of a rotational assemblyaccording to one embodiment of the present invention.

FIG. 6 illustrates a partial cross-section view of a rotational assemblyaccording to one embodiment of the present invention.

FIG. 7 illustrates a perspective view of a rotational assembly accordingto one embodiment of the present invention.

FIG. 8 illustrates a cross-section view of a rotational assemblyaccording to one embodiment of the present invention.

FIG. 9 illustrates a cross-section view of a rotational assemblyaccording to one embodiment of the present invention.

FIG. 10 illustrates a cross-section view of an impeller according to oneembodiment of the present invention.

FIG. 11 illustrates a partial cross-section view of an impelleraccording to one embodiment of the present invention.

FIG. 12 illustrates a perspective view of drive shaft core according toone embodiment of the present invention.

FIG. 13 illustrates a cross-section view of an impeller according to oneembodiment of the present invention.

FIG. 14A illustrates a perspective view of an impeller according to oneembodiment of the present invention.

FIG. 14B illustrates a perspective view of an impeller according to oneembodiment of the present invention.

FIG. 15 illustrates a cross-section view of a sparger body according toone embodiment of the present invention.

FIG. 16 illustrates a cross-section view of a sparger assembly accordingto one embodiment of the present invention.

FIG. 18 illustrates a cross-section view of a sparger assembly accordingto one embodiment of the present invention.

FIG. 19 illustrates a cross-section view of a sparger assembly accordingto one embodiment of the present invention.

FIG. 20 illustrates a partial perspective view of a reactor systemaccording to one embodiment of the present invention.

FIG. 21 illustrates a partial perspective view of a reactor systemaccording to one embodiment of the present invention.

FIG. 22 illustrates a partial perspective view of a reactor systemaccording to one embodiment of the present invention.

FIG. 23 illustrates a cross-section view of a reactor system accordingto one embodiment of the present invention.

FIG. 24 illustrates a perspective view of a reactor system according toone embodiment of the present invention.

FIG. 25 illustrates a perspective view of a reactor system according toone embodiment of the present invention.

FIG. 26 illustrates a probe assembly 2600 according to one embodiment ofthe present invention.

FIG. 27A provides an illustration of a probe port subassembly of a probeassembly according to one embodiment of the present invention.

FIG. 27B illustrates a probe kit subassembly of a probe assemblyaccording to one embodiment of the present invention.

FIG. 27C illustrates an autoclave subassembly of a probe assemblyaccording to one embodiment of the present invention.

FIG. 28A illustrates a probe assembly according to one embodiment of thepresent invention.

FIG. 28B illustrates a probe assembly according to one embodiment of thepresent invention.

FIG. 29 provides a graph of data that was generated using a reactorsystem according to one embodiment of the present invention.

FIG. 30 provides a graph of data that was generated using a reactorsystem according to one embodiment of the present invention.

FIG. 31 provides a graph of data that was generated using a reactorsystem according to one embodiment of the present invention.

FIG. 32 provides a graph of data that was generated using a reactorsystem according to one embodiment of the present invention.

FIG. 33 provides a graph of data that was generated using a reactorsystem according to one embodiment of the present invention.

FIG. 34 provides a graph of data that was generated using a reactorsystem according to one embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments, the term “flexible bag” can refer to a containerthat holds a fluidic medium. The bag may include one or more layer(s) offlexible or semi-flexible waterproof material depending on size,strength and volume requirements. The inside surface of the bag may besmooth and provide a sterile environment (e.g., for culturing cells orother organisms, for food production, etc.). The bag may include one ormore openings, pouches (e.g., for inserting one or more probes, devices,etc.), ports (e.g., for the connection of one or more probes, devices,etc.) or the like. Furthermore, the bag can provide a disposablealternative to a solid vessel in a conventional stirred-tank bioreactor.The flexible bag may further include a shaft, an impeller, a bearing andseals or O-rings, and may be entirely disposable.

In some embodiments, the term “fluidic medium” can refer to anybiological fluid, cell culture medium, tissue culture medium, culture ofmicroorganisms, culture of plant metabolites, food production, chemicalproduction, biopharmaceutical production, and the like. The fluidicmedium is not limited to any particular consistency and its viscositymay vary from high to medium to low. When the fluidic medium is a cellculture medium the system may be operated in, for example, batch mode,semi-batch mode, fed-batch mode, or continuous mode.

In some embodiments, the term “impeller” can refer to a device that isused for agitating or mixing the contents of a stirred-tank reactorsystem (e.g., bioreactor). The impeller may agitate the fluidic mediumby stirring or other mechanical motion. The impeller of the instantinvention includes, but is not limited to, a Rushton, a marine, ahydrofoil, a pitched blade, and any other commercially availableimpeller.

In some embodiments, a “hydrodynamic” environment of the instantinvention may refer to an environment that is influenced by the motionof fluids and the forces acting on solid bodies immersed in these fluidswithin the stirred-tank reactor system.

The present invention includes single use bioreactors, stirred tankreactors, and the like. Such reactors have a variety of applications,such as for the production of therapeutic proteins via batch cellculture. Relatedly, these systems can be used to provide for cell growthand antibody production for CHO and other cell lines. The hydrodynamicenvironment within the reactors can be well characterized, and, as such,may be scaled to other stirred tank bioreactors.

Single use bioprocess containers can be used for the storage ofbiopharmaceutical media, buffers, and other products. Using thesestorage container systems, several mixing systems for preparation ofmedia and buffers can be developed, often to commercial scale up to10,000 liters or more. Such mixing systems and bioreactors can usevarious means for mixing the reactor contents, such as a pulsating disk,a paddle mixer, a rocking platform, an impeller, and the like. Thesesystems are well suited for use in chemical processing. The operatingcharacteristics of the reactors can be well defined, and can be readilypredicted and scaled to various sizes. In the biopharmaceuticalindustry, such stirred tank bioreactors can be established as a meansfor manufacture of biologic products from a wide range of biologicalsystems, including animal cell culture. Processes for biological systemscan be developed using stirred tank bioreactors at the bench scale andtransferred to stirred tank bioreactors at the commercial scale, up to10,000 liters or greater, using well established scale-up methodologies.For a stirred tank bioreactor, design parameters such as tip speed,power input, Reynolds number, and oxygen transfer coefficient can bereadily determined and used for scale-up.

A single use portion of the system can include a flexible plasticcontainer with the following single use integrated components: abearing, shaft, and impeller assembly; a sparger assembly; ports forsterile attachment of sensor probes; and various ports for inlet andoutlet of liquids and gases. A single use bioreactor can be manufacturedusing medical grade film. In some cases, other components of the singleuse bioreactor can be manufactured from readily machined materials thatare not necessarily USP Class VI materials. The impeller can be apitched-blade impeller that is attached to a bearing assembly by aflexible sheath. The impeller and sheath can rotate along with an innerbearing assembly, which is isolated from the exterior bearing assemblyusing various seal assemblies. An outer bearing assembly can be directlyaffixed to the single use container. A sparger can include a porousmembrane that is sealed to the bottom of the single use container.Sparge gas can be introduced to the space between the porous membraneand bottom of the container through a port after passing through apre-attached sterilization filter. The pH and dO₂ sensors may or may notbe part of the single use container and can be connected to thebioreactor using PALL KLEENPACK® connectors. Industry-standard 12 mmsensors can be calibrated, then steam sterilized with one half of theconnector. The other half of the connector can be pre-attached to thecontainer, allowing the sensor to be inserted in direct contact with thereactor contents. Ports and tubing for headspace gas, thermo well, mediainlet, titrant, sampling, harvest, and various pulse feeds can bepre-attached and pre-sterilized with the container.

A permanent support vessel that contains a motor and drive shaftassembly, heat jacket, and openings for inlets, outlets, and probes canhold a single use container. A drive shaft can fit through thesingle-use bearing, through the flexible sheath, and lock into theimpeller. This shaft can be driven using a standard bioreactor mixermotor of sufficient power. Heat can be provided to the bioreactorcontents, for example, by electric heat bands that are in direct contactwith sides of the single-use container. The permanent support vessel canbe mobile, and can be placed on a weigh scale for control of reactorvolume.

The system can be operated using standard sensors and controllers thathave industry-accepted track records of performance. In someembodiments, no control system may be required for steam sterilizationor clean in place, and a controller commonly used for bench-scalebioreactors may be sufficient for control of the pH, dO₂ concentration,and temperature of the single use bioreactor. A single use bioreactoroften requires no cleaning or sterilization in-place. As such, thecapital and operating costs of control systems and utilities, such asclean steam, required for steam sterilization of a large pressure vesselmay be eliminated. The cost for fabrication of a rigid-walled pressurevessel designed to handle the stresses exerted during steam-in-placesterilization may also be eliminated. Likewise, the capital andoperating costs for clean-in-place control systems and utilities may beunnecessary. The design elements of traditional stainless steel vesselsdictated by cleanability requirements may similarly be eliminated.

In some embodiments, a single use bioreactor can be a closed system thatis discarded after use. This may eliminate the need for cleaningvalidation studies. The potential for cross contamination betweenproduction batches may also be reduced. In some embodiments, the capitalexpenditure required to accommodate multiple products simultaneously insingle use bioreactors can be low compared to the cost of the fixedassets and utilities required to segregate traditional bioreactorsystems. A single use bioreactor can be manufactured using medical gradefilm, and regulatory documentation for the film may be currentlyavailable. Other product contact components of a single use bioreactorcan be manufactured from USP Class VI materials. Current applications ofbioprocess containers manufactured from these materials includebioreactor feed and harvest, and transport and storage of bulkintermediate and final product.

As noted above, a stirred tank single use bioreactor according to thepresent invention can provide a well-characterized hydrodynamicenvironment for cell growth. Mixing characteristics can be readilycalculated and can be translated to larger stirred tank reactors. Thus,processes developed at the lab or pilot scale may be scaled up directlyto commercial scale, either in larger single use bioreactors or largertraditional stirred tank bioreactors. Scale-up parameters such as powerinput per unit volume, tip speed, oxygen transfer coefficient, orgeometric similarity may be maintained at the larger scale. In someembodiments, the present invention provides a stirred tank reactor witha design that includes a rotating impeller driven by a drive shaftisolated through a series of rotating seals. Such designs can provideeffective and efficient means of transmitting the energy required formixing and mass transfer to the reactor contents.

The present invention can also include or be compatible withindustry-standard sensor and controller technology. A standard that hasdeveloped in the industry is the use of 12 mm diameter pH and dO₂sensors inserted through DN25 (Inglold-style) ports in direct e contactwith the reactor contents. Systems such as a single use bioreactor canincorporate the same 12 mm diameter pH and dO₂ sensors in direct contactwith the reactor contents. Calibration and standardization proceduresfor these sensors can be readily performed during operation of thebioreactor. In addition, outputs from these sensors can be compatiblewith current controllers used by industry. The use of PID controllers tomaintain pH, dO₂ concentration, and temperature can be used in suchbioreactors. As a stirred tank bioreactor with standard sensors, thesecontrol strategies can be directly translatable to a single usebioreactor. Because it can be a stand-alone unit, the single usebioreactor may be controlled using whichever controller type that ispreferred by a given facility.

A. The Stirred-Tank Reactor System

In some embodiments, the stirred-tank reactor system of the presentinvention provides a flexible and disposable bag for a variety ofpurposes, including culturing cells, microorganisms, or plantmetabolites as well as processing foods, chemicals, biopharmaceuticalsand biologicals. The disposable bag may include disposable elements suchas a shaft, impeller and bearing and is designed to fit into a permanenthousing such as a reactor housing. The bag may further include one ormore openings, pouches, ports or the like. The stirred-tank reactorsystem allows a user to operate the culture or production with relativeease and little training. In particular, the disposable system may notrequire cleaning or sterilizing. Furthermore, the system may not needcontinuous validation between production runs. Thus, it combines aflexible, easy-to-use, true stirred-tank reactor environment with littleor no cross-contamination during the production process.

Referring to the drawings, FIG. 1 depicts a flexible bag 104 with atleast one opening and an agitation shaft 112 with an attachable impeller113. As shown, the agitation shaft 112 and attached impeller 113 aresituated within the bag 104. Further, the agitation shaft 112 isconnectable to a bearing 105, wherein the bearing 105 can be sealed tothe bag by heat welding to the bag and/or through seal(s) or O-ring(s)106. The bag 104, agitation shaft 112, impeller 113, and bearing 105,including seals or O-rings 106 are optionally disposable. The disposablebag can be a flexible, plastic bag. The bag 104 can be affixed to theagitation shaft 112 and the bearing 105 through at least one seal orO-ring 106 such that the inside of the bag remains sterile. The seals oro-rings can be further affixed to the bag as is shown in FIG. 1 .Additionally, the disposable stirred-tank reactor system may beconnected to a support or one or more bracket(s) 103 that hold thebearing 105 and motor 101. In one embodiment (as shown in FIG. 1 ), thesupport 103 is a motor and bearing support 103, wherein the upper end ofthe agitation shaft 112 is further connected to a motor coupling 102.The motor coupling 102 is connected to the motor 101 which drives thestirring motion of the agitation shaft 112 and impeller 113 leading to ahydrodynamic environment within the bag 104. The bag 104 is designed tofit into a housing 111 such as a barrel or chamber. The housing may be ametal barrel, a plastic barrel, a wood barrel, a glass barrel, or anyother barrel or chamber made from a solid material. In one embodiment ofthe instant invention, the housing further includes a plurality ofbaffles, wherein the bag folds around the baffles. In anotherembodiment, the flexible bag 104 further includes a top port (single ormultiple) 108, a bottom port (single or multiple) 109, and a side port(single or multiple) 110, wherein flexible tubing 107 can be connectedto one or more of these ports.

The stirred-tank reactor system may optionally include a heater such asa heating pad, a steam jacket, or a circulating fluid or water heater.In one embodiment, the heater is located between the bag 104 and thehousing 111. In another embodiment, the heater is incorporated into thehousing 111 (e.g., into a double wall between the reactor housing andthe bag). In yet another embodiment, the stirred-tank reactor system isplaced inside an incubator. The heater allows for heating or warming ofa specific culture or production. This is particularly important forcell cultures which are often grown at 37° C.

In one embodiment of the instant invention, the bag 104, the bearing105, the seal(s) or O-ring(s) 106, the tubing 107, the top port(s) 108,the bottom port(s) 109, the side port(s) 110, the shaft 112, and theimpeller 113 are disposable. The motor 101, the motor coupling 102, thebracket(s) or motor and bearing support 103, and the housing 111 arepermanent.

B. Devices and Ports

The stirred-tank reactor system may also include sensors and otherdevices. In one embodiment, the bag includes a pH sensor and adissolved-oxygen sensor, wherein the sensors are incorporated into thebag. As such, the sensors are disposable with the bag. In anotherembodiment, the sensors are attachable to the bag and are separateunits. Such sensors may optionally be reusable after sterilization. Inanother embodiment, the system includes a product loop with flow past apH sensor and dissolved-oxygen sensor, wherein the sensors areincorporated into the reactor housing. The system is flexible andprovides alternative ways of supplying optional equipment of variouskinds (e.g., sensors, probes, devices, pouches, ports, etc.). The systemmay also include one or more internal pouches that are sealed to thebag. In one preferred embodiment, the pouch has at least one end thatcan be opened to the outside of the bag to insert a probe into thereactor (i.e., the bag) while remaining on the exterior of the bag. Theprobe may be, for example, a temperature probe, a pH probe, a dissolvedgas sensor, an oxygen sensor, a carbon dioxide sensor, a cell masssensor, a nutrient sensor, an osmometer or any other probe that allowsfor testing or checking the culture or production. In another preferredembodiment, the system includes at least one port in the bag allowingfor the connection of a device to the port. Such a device includes, butis not limited to, a tube, a filter, a connector, a probe, and asampler. The incorporation of various ports into the bag allows for gasflow in and out of the bag as well as liquid flow in and out of the bag.Such ports also allow for sampling or testing the media or cultureinside the bag. Tubing, filters, connectors, probes, samplers or otherdevices can be connected to the ports by using any desirable tubingconnection technology. Pouches and ports that are sealed or affixed tothe bag are disposable with the bag. The bag may also include a sparger(i.e., the component of a reactor that sprays air into the medium)sealed to the bag which can be disposed off with the bag.

Particularly, ports may be incorporated at any place on the flexible bagto accommodate the following:

Headspace gas in

Headspace gas out

Sparge gas in

Temperature probe

pH probe

Dissolved oxygen probe

Other desired probes

Sample apparatus

Media in

Titrant in

Inoculum in

Nutrient feeds in

Harvest out

Each port may have flexible tubing attached to the port, to which mediabags, sample devices, filters, gas lines, or harvest pumps may beattached with sterile or aseptic connections. In one embodiment, theports are sealed onto the flexible bag during bag manufacture, and aresterilized with the bag assembly.

Devices that may be used to make aseptic connections to the flexibletubing are the following:

-   -   WAVE sterile tube fuser    -   TERUMO sterile tubing welder    -   PALL KLEENPAK® connector    -   Connection made under a laminar flow hood, using aseptic        techniques    -   BAXTER Hayward proprietary “HEAT-TO-HEAT” connection using metal        tubing and an induction heater

In another embodiment, flexible tubing that is attached to anappropriate stainless-steel valve assembly may be sterilized separately(e.g., via autoclave), and then used as a way to connect the disposablebioreactor to traditional reactors or process piping. The valve assemblyis used to make a traditional steam-in-place (SIP) connection to atraditional reactor or other process, and the flexible tubing is used tomake a sterile or aseptic connection to a port on the disposablereactor.

Referring to the drawings, FIG. 2 depicts a probe connection that can beemployed with the stirred-tank reactor system according to oneembodiment of the instant invention. As shown in FIG. 2 , the probe 201can be connected to a flexible sleeve 202 or bag which extends to onehalf of a PALL connector 203. The PALL connector 203 can be connected tothe other half of the PALL connector 205 to provide for a sterileconnection between the probe and the stirred-tank reactor system. ThePALL connectors 203, 205 include covers 204 and filters 207 to keep theconnection site sterile. Filters 207 can comprise a sealing layer orstripout layer. Sterile tubing 206 extends from the other half of thePALL connector 205 to a reactor port 208 of the reactor vessel 209 ofthe stirred-tank reactor system. In order to attach the probe, the PALLconnection is made by removing the covers 204, mating the connectors203, 205, removing the filters 207, and sliding the movable part of theconnector into position. The probe sensor tip 212 is then pushed intothe reactor as the flexible sleeve or bag bunches or compresses 210. Theprobe sensor tip 212 is then in direct contact with the inside of thereactor vessel 209. A clamp 211 is placed around the probe and tubing toseal the reactor contents from the PALL connection assembly. Thus, whena sterile connection is made between the two halves of the PALLconnectors 203, 205, the flexible sleeve 202 or bag becomes compressed210 and the probe is in contact with the culture or production media.

In one embodiment, the probes may be sterilized separately (e.g., viaautoclave) then attached to the reactor via a sterile or asepticconnection. For example, a probe assembly may be made by inserting aprobe 201 into one half of a PALL KLEENPAK® connector 203 and sealingthe probe to the connector using a flexible sleeve or bag 202 asdescribed above and shown in FIG. 2 . The sleeve extends from theoutside end of the probe to the barb of the PALL connector. Thisassembly is sterilized separately. The other half of the PALL connector205 is connected to a port 208 on the reactor 209 via flexible tubing206 that will accommodate the probe. This assembly is sterilized as partof the reactor. The PALL connector is described in detail in U.S. Pat.No. 6,655,655, the content of which is incorporated herein by referencein its entirety.

FIGS. 3A and 3B illustrate cross-section views of a reactor system 300according to one embodiment of the present invention. Reactor system 300can include a rotational assembly 301 coupled with a container 302.Optionally, reactor system 300 may include an impeller 340. In someembodiments, rotational assembly 301 is in sealed cooperation with anopening or aperture in container 302. Similarly, rotational assembly 301may include a casing 360 that is coupled with the opening or aperture incontainer 302. Typically, impeller 340 is disposed within the interiorof container 302. Rotational assembly 301 can be supported or held bybracket 308.

In some embodiments, rotational assembly 301 may include a hub 320 thatis coupled with impeller 340, and hub 320 may be coupled with impeller340 via a connector 390. Optionally, hub 320 may be directly coupledwith impeller 340. In some embodiments, hub 320 is tubular in shape andincludes an interior surface which bounds a passageway 320 alongitudinally extending therethrough. In one embodiment an annular barb321 radially encircles and outwardly projects from the exterior surfaceof hub 320. Barb 321 can be used for creating a sealed connection withconnector 390.

Connector 390 can be tubular in shape, and can include an interiorsurface which bounds a passageway 390 a extending longitudinallytherethrough. In some embodiments, connector 390 includes a flexibletube having a first end connected in sealed engagement with hub 320 andan opposing second end connected in sealed engagement with impeller 340.Hub 320, either alone or in cooperation with connector 390, can providea sealed channel in which drive shaft 304 can be received and removablycoupled with impeller 340. Consequently, drive shaft 304 can be usedrepeatedly without sterilizing because it does not directly contact thecontents of container 302. Furthermore, by using a flexible tube asconnector 390, a flexible container 302 such as a bag assembly can beeasily rolled up or folded for easy transport, storage, or processing.

Often, rotational assembly 301 will include a bearing assembly 370disposed between hub 320 and casing 360. Bearing assembly 370 caninclude a journal bearing, which may be in fixed relation with casing360, and hub 320 can rotate relative to the journal bearing and casing360. Hub 320 may include a guide 324 for receiving a snap ring orretaining ring, which can help maintain hub 320 in place, relative tothe journal bearing.

Rotational assembly 301 may also include a sealing arrangement 380,which can be disposed between hub 320 and casing 360. Sealingarrangement 380 can include, for example, a wear plate 382 and one ormore seals 384, which may be, for example, dynamic seals. Wear plate 382can be disposed circumferentially to, and coupled with, hub 320. Seal(s)384 can be disposed between wear plate 382 and casing 360. Rotationalassembly 301 may also include one or more seals 392 disposed betweenwear plate 382 and hub 320, wherein seals 392 may be, for example,static seals. In some embodiments, seal(s) 384 include one or moreV-rings and seals(s) 392 include one or more O-rings. In the embodimentsshown in FIG. 3A, seal(s) 384 include two V-rings, and seal(s) 392include one O-ring. An annular flange 323 may also radially, outwardlyproject from the exterior surface of hub 320 and be disposed againstseal 392.

In use, hub 320 is configured to receive or house a drive shaft 304 thatis selectively coupled with a motor (not shown). In some embodiments,hub 320 may be configured to couple with one or more ears 306 located atan upper end of drive shaft 304 via one or more hub notches 322 formedon hub 320. Impeller 340 may include a spline 342 configured to couplewith a lower end of drive shaft 304. Drive shaft 304 can be placed inhub 320, and coupled with hub 320 and impeller 340. For example, driveshaft 304 may extend through passageway 320 a. Similarly, drive shaft304 may extend through passageway 390 a. Drive shaft 304 can be rotatedby a motor, thereby rotating hub 320, connector 390, and impeller 340.In turn, impeller 340 agitates the contents of container 302. As hub 320is rotated by drive shaft 304, seal(s) 392 provide a seal between wearplate 382 and hub 320 as they both rotate in unison, relative to casing360. As casing 360 remains stationary, seal(s) 384 provide a sealbetween wear plate 382 and casing 360, where wear plate 382 rotatesrelative to casing 360. In some embodiments, seal(s) 384 provide ahermetic seal between wear plate 382 and casing 360. As shown here,seal(s) 384 can be in co-planar arrangement with one another.

In some embodiments, hub 320 may be removably engagable with drive shaft304 such that annular rotation of drive shaft 304 facilitates annularrotation of hub 320. Although the embodiment depicted in FIG. 3A showsdrive shaft ears 306 coupled with hub notches 322, the present inventioncontemplates any of a variety of coupling means for accomplishing thisfunction. In yet other alternative embodiments, clamps, pins, collets,meshing teeth, or other fasteners can be used to removably secure driveshaft 304 to the hub 320 when the drive shaft 304 is coupled with hub320. Similarly, the present invention contemplates any of a variety ofcoupling means for removably engaging drive shaft 304 to impeller 340,including the coupling means described above, such that rotation ofdrive shaft 304 facilitates rotation of impeller 340.

According to one embodiment of the present invention, reactor system 300can be manufactured by coupling container 302 with rotational assembly301, such that container 302 and rotational assembly 301 are in sealedcooperation with one another. For example, rotational assembly 301 canbe coupled with an opening of container 302. Rotational assembly 301 canbe manufactured to include hub 320, and hub 320 can be coupled withimpeller 340 such that impeller 340 is disposed within container 302.Further, reactor system can be sterilized, for example by gammaradiation.

According to another embodiment of the present invention, reactor system300 can be prepared for use by coupling casing 360 of rotationalassembly 301 to frame bracket 308, and placing container 302 at leastpartially within a frame or container housing (not shown). Drive shaft304 can be inserted into hub 320, and a distal end of drive shaft 304can be coupled with impeller 340. Further, reaction components such ascells and culture media can be introduced into container 302 via a port310.

Container 302 can include any of a variety of materials. In someembodiments, container 302 includes a flexible bag of water impermeablematerial such as a low-density polyethylene or other polymeric sheetshaving a thickness in a range between about 0.1 mm to about 5 mm, orbetween about 0.2 mm to about 2 mm. Other thicknesses can also be used.The material can be comprised of a single ply material or can comprisetwo or more layers which are either sealed together or separated to forma double wall container. Where the layers are sealed together, thematerial can comprise a laminated or extruded material. The laminatedmaterial can include two or more separately formed layers that aresubsequently secured together by an adhesive. The extruded material caninclude a single integral sheet having two or more layers of differentmaterial that are each separated by a contact layer. All of the layerscan be simultaneously co-extruded. One example of an extruded materialthat can be used in the present invention is the HyQ CX3-9 filmavailable from HyClone Laboratories, Inc. out of Logan, Utah. The HyQCX3-9 film is a three-layer, 9 mil cast film produced in a cGMPfacility. The outer layer is a polyester elastomer coextruded with anultra-low density polyethylene product contact layer. Another example ofan extruded material that can be used in the present invention is theHyQ CX5-14 cast film also available from HyClone Laboratories, Inc. TheHyQ CX5-14 cast film comprises a polyester elastomer outer layer, anultra-low density polyethylene contact layer, and an EVOH barrier layerdisposed therebetween. In another example, a multi-web film producedfrom three independent webs of blown film can be used. The two innerwebs are each a 4 mil monolayer polyethylene film (which is referred toby HyClone as the HyQ BM1 film) while the outer barrier web is a 5.5 milthick 6-layer coextrusion film (which is referred to by HyClone as theHyQ BX6 film).

FIG. 4A illustrates a cross-section view of a rotational assembly 401according to one embodiment of the present invention. FIG. 4Billustrates a cross-section view of the D rotational assembly 401depicted in FIG. 4A coupled with a connector 490 and an impeller 440.Rotational assembly 401 may include a bearing assembly 470 disposedbetween a hub 420 and a casing 460. As shown here, bearing assembly 470includes two race bearings, which are in fixed relation with casing 460.Hub 420 can rotate relative to the race bearings. Hub 420 may includeguides 424, 424 a for receiving a snap ring or retaining ring, which canhelp maintain hub 420 in place, relative to race bearings.

Rotational assembly 401 may also include a sealing arrangement 480,which can be disposed between hub 420 and casing 460. Sealingarrangement 480 can include, for example, a wear plate 482, one or moreseals 484, and a rotating disk 450. Rotating disk 450 can be disposedcircumferentially to, and coupled with, hub 420. Seal(s) 484 can bedisposed between rotating disk 450 and wear plate 482. Wear plate 482can be coupled with casing 460 via screws or bolts inserted throughcasing columns 428. Rotational assembly 401 may also include one or moreseals 492 disposed between rotating disk 450 and hub 420. In someembodiments, seal(s) 484 include one or more V-rings and seals(s) 492include one or more O-rings. In the embodiment shown in FIGS. 4A and 4B,seal(s) 484 include three V-rings, and seal(s) 492 include one O-ring.Rotational assembly 401 may also include one or more seals 426 toprovide a seal between hub 420 and the top of casing 460, and one ormore seals 462 to provide a seal between casing 460 and wear plate 482.As shown here, seal(s) 426 include one V-ring and seal(s) 462 includeone O-ring.

In use, hub 420 is configured to receive or house a drive shaft (notshown). In some embodiments, hub 420 may be configured to couple with anear of drive shaft via hub notch 422. As hub 420 is rotated by driveshaft, seal(s) 492 provide a seal between rotating disk 450 and hub 420as they both rotate in unison, relative to casing 460. As casing 460remains stationary, seal(s) 484 provide a seal between rotating disk 450and wear plate 482, where rotating disk 450 rotates relative to wearplate 482 and casing 460. In some embodiments, seal(s) 484 provide ahermetic seal between rotating disk 450 and wear plate 482. As shownhere, seal(s) 484 can be in co-planar arrangement with one another.

FIG. 5 illustrates a cross-section view of a rotational assembly 501according to one embodiment of the present invention. Rotationalassembly 501 may include a bearing assembly 570 disposed between a hub520 and an inner casing 560. As shown here, bearing assembly 570includes two race bearings, which are in fixed relation with innercasing 560. Hub 520 can rotate relative to the race bearings. Hub 520may include guides 524, 524 a for receiving snap rings or retainingrings, which can help maintain hub 520 in place, relative to racebearings.

Rotational assembly 501 may also include a sealing arrangement 580.Sealing arrangement 580 can include, for example, a bottom plate 583 andone or more seals 584. Seal(s) 584 can be disposed between hub 520 andinner casing 560. A top plate 587 can be coupled with inner casing 560via screws or bolts inserted through casing columns 528. Rotationalassembly 501 may also include one or more seals 591 disposed between topplate 587 and an outer casing 561. In some embodiments, seal(s) 584include one or more V-rings and seals(s) 591 include one or moreO-rings. In the embodiment shown in FIG. 5 , seal(s) 584 include threeV-rings, and seal(s) 591 include one O-ring. Rotational assembly 501 mayalso include one or more seals 526 to provide a seal between hub 520 andthe top plate 587. As shown here, seal(s) 526 include one V-ring.

In use, hub 520 is configured to receive or house, and couple with, adrive shaft (not shown). As hub 520 is rotated by drive shaft, seal(s)584 provide a seal between hub 520 and inner casing 560 as hub 520rotates relative to inner casing 560. In some embodiments, seal(s) 584provide a hermetic seal between hub 520 and inner casing 560. As shownhere, seal(s) 584 can be in co-planar arrangement with one another.

FIG. 6 illustrates a partial cross-section view of a rotational assembly601 according to one embodiment of the present invention. Rotationalassembly 601 may include a bearing assembly 670 disposed between a hub620 and an inner casing 660. As shown here, a lower race bearing of thebearing assembly 670 is in fixed relation with inner casing 660. Hub 620can rotate relative to the race bearing. Hub 620 may include a guide 624a for receiving snap rings or retaining rings, which can help maintainhub 620 in place, relative to race bearing.

Rotational assembly 601 may also include a sealing arrangement 680.Sealing arrangement 680 can include, for example, one or more seals 684.Seal(s) 684 can be disposed between hub 620 and inner casing 660. Insome embodiments, seal(s) 684 include one or more V-rings. In theembodiment shown in FIG. 6 , seal(s) 684 include three V-rings.

In use, hub 620 is configured to receive or house, and couple with, adrive shaft (not shown). As hub 620 is rotated by drive shaft, seal(s)684 provide a seal between hub 620 and inner casing 660, as hub 620rotates relative to inner casing 660. In some embodiments, seal(s) 684provide a hermetic seal between hub 620 and inner casing 660. As shownhere, seal(s) 684 can be in a tiered-planar arrangement with oneanother.

FIG. 7 illustrates a perspective view of a rotational assembly 701according to one embodiment of the present invention. Rotationalassembly 701 can include a hub 720 having one or more hub notches 722.In use, hub 720 is configured to receive or house, and couple with, adrive shaft 704. Hub notch(es) 722 are configured to couple with one ormore drive shaft ears 706. A top plate 787 can be coupled with casing760 via screws or bolts inserted through top plate apertures 787 a. Ashub 720 is rotated by drive shaft 704, hub 720 rotates relative to topplate 787 and casing 760. Rotational assembly 701 may also include oneor more seals 726 to provide a seal between hub 720 and the top plate787. As shown here, seal(s) 726 include one V-ring.

FIG. 8 illustrates a cross-section view of a rotational assembly 801according to one embodiment of the present invention. Rotationalassembly 801 can include a hub 820 having one or more hub notches 822.As shown here, a bearing assembly 870 is in fixed relation with ahousing 823. In use, hub 820 is configured to receive or house, andcouple with, a drive shaft 804. Hub notch(es) 822 are configured tocouple with one or more drive shaft ears 806, which may be at opposingends of a drive shaft spindle 806 a. As hub 820 is rotated by driveshaft 804, hub 820 rotates relative to housing 823, bearing assembly870, and casing 860.

Rotational assembly 801 may also include a sealing arrangement 880,which can be disposed between hub 820 and housing 823. Sealingarrangement 880 can include, for example, one or more outer seals 884and one or more inner seals 886. Seal(s) 884 can be disposed between anouter surface of hub cup 820 a and housing 823, and seal(s) 886 can bedisposed between an inner surface of hub cup 820 a and housing 823.Housing 823 can be fixed with casing 860. In some embodiments, seal(s)884 include one or more V-rings and seals(s) 886 include one or more oilseals. In the embodiment shown in FIG. 8 , seal(s) 884 include oneV-ring, and seal(s) 886 include one oil seal. Hub 820 can be coupledwith a flexible tube 890.

FIG. 9 illustrates a cross-section view of a rotational assembly 901according to one embodiment of the present invention. Rotationalassembly 901 can include a hub 920 configured to releasably couple witha drive shaft 904. As shown here, two bearings of a bearing assembly 970are in fixed relation with a housing 923. In use, hub 920 is configuredto receive or house, and couple with, a drive shaft 904. As hub 920 isrotated by drive shaft 904, hub 920 rotates relative to housing 923,bearing assembly 970, and casing 960.

Rotational assembly 901 may also include a sealing arrangement 980,which can be disposed between hub 920 and inner housing 923 a. Sealingarrangement 980 can include, for example, one or more outer seals 984and one or more inner seals 986. Seal(s) 984 can be disposed between hub920 and seal(s) 986, and seal(s) 986 can be disposed between seal(s) 984and inner housing 923 a. Housing 923 can be fixed with casing 960, andin sealed relation with casing 960 via one or more seal(s) 962. In someembodiments, seal(s) 984 include one or more V-rings, seals(s) 986include one or more oil seals, and seal(s) 962 include one or moreO-rings. In the embodiment shown in FIG. 9 , seal(s) 984 include twoV-rings, seal(s) 986 include two oil seals, and seal(s) 962 include twoO-rings. Hub 920 can be coupled with a flexible tube 990.

FIG. 10 illustrates a cross-section view of an impeller 1040 accordingto one embodiment of the present invention. Impeller 1040 can be coupledwith connector 1090, which can be coupled with hub (not shown). Impeller1040 can include an impeller spline 1042 which can couple with a spline1005 of drive shaft 1004.

FIG. 11 illustrates a partial cross-section view of an impeller 1140according to one embodiment of the present invention. Impeller 1140 caninclude an impeller barb fitting 1141 that can couple with a rotationalassembly hub (not shown) via a connector 1190. Drive shaft 1104 can beattached to impeller 1140 by placing drive shaft 1104 into impelleraperture 1142. When drive shaft 1104 is inserted into impeller aperture1142, end cap 1107 can reach the distal end of impeller base 1143. Asshown here, drive shaft 1104 is hollow and adapted to receive a core1108. Drive shaft 1104 is coupled with an end cap 1107. Core 1108includes a ball dent 1102 which operatively associates with a ball 1103.In a first ball configuration 1103 a, ball 1103 is disposed at ball dent1102. As core 1108 is advanced along the inside of hollow drive shaft1104 toward the distal end of impeller aperture 1142, spring 1109 iscompressed, and ball 1103 moves into opening 1104 a in drive shaftopening 1104 a and impeller base opening 1143 a, thus adopting a secondball configuration 1103 b. Distal end of core 1108 can cause end cap1107 to separate from drive shaft 1104. In some embodiments, core 1108is in threaded engagement with end cap 1107, which can prevent spring1109 from pushing core 1108 back out of hollow drive shaft 1104.

FIG. 12 illustrates a perspective view of drive shaft core 1208according to one embodiment of the present invention. Drive shaft core1208 includes ball dent 1202, end cap 1207, spring 1209, and ball 1203.As shown here, ball 1203 can adopt a first ball configuration 1203 a anda second ball configuration 1203 b.

FIG. 13 illustrates a cross-section view of an impeller 1340 accordingto one embodiment of the present invention. Impeller 1340 can include asquare spline 1342 for coupling with a square spline 1305 of drive shaft1304. Impeller 1340 can be coupled with hub (not shown) via a connector1390. For the sake of clarity, the impeller blades are not shown in thisfigure.

FIG. 14A illustrates a perspective view of an impeller 1440 a accordingto one embodiment of the present invention. Impeller 1440 a can includeone or more impeller blades 1445 a coupled with an impeller body 1446 a.In some embodiments, impeller blades 1445 a can be machined separatelyfrom impeller body 1446 a. Impeller blades 1445 a may be constructedfrom a variety of materials, including Delrin, HDPE, and the like.Impeller body 1446 a may be constructed from a variety of materials,including HDPE and the like.

FIG. 14B illustrates a perspective view of an impeller 1440 b accordingto one embodiment of the present invention. Impeller 1440 b can includeone or more impeller blades 1445 b and an impeller body 1446 b. In someembodiments, impeller 1440 b can be molded as a single piece. Impeller1440 b may be constructed from a variety of materials, including mediumlow density polyethylene, low density polyethylene, DOW ENGAGE®polyolefin elastomers, and the like.

FIG. 15 illustrates a cross-section view of a sparger body 1500according to one embodiment of the present invention. Sparger body 1500can include a sheet of permeable material. In some embodiments, spargerbody 1500 includes a vapor-permeable and water-resistant material. Inrelated embodiments, sparger body 1500 includes a high densitypolyethylene fiber. For example, sparger body 1500 can include TYVEK®material. Sparger body 1500 can be in fluid communication with a port ofa container (not shown) via a sparger conduit 1510. As shown in FIG. 15, sparger body 1500 can be in the shape of a donut or ring. Relatedly,sparger body 1500 can include a base 1502 which is adapted to anchor toan interior surface of a container (not shown). The base may or may notinclude a gas permeable material. In other embodiments, one or moresheets of gas permeable material can be directly sealed with theinterior of the container, whereby the interior of the sparger body 1500includes a gas permeable material on one side (e.g. top side of body),and a corresponding portion of the container on the other side (e.g.bottom side of body).

In some embodiments, the permeability of the sparger body is such thatfluid is prevented from flowing into the sparger when not in use.Similarly, the sparger may be constructed so as to only allow gas topass through the permeable material when it is subject to sufficientlyhigh gas pressure. Often, a sparger body will include a soft, flexiblematerial. In some embodiments, sparger body 1500 may be welded directlyonto the container so as to ensure proper placement and alignment. Whencoupled with a flexible container such as a flexible bag, sparger body1500 can effectively be folded up with the bag for storage andtransport, sterilized simultaneously with the bag, and disposed of so asto eliminate subsequent cleaning. Sparger body 1500 can provide forminute gas bubbles which can increase diffusion of gas into the fluid.It is appreciated that other types of spargers can be used with thepresent system.

A variety of materials or assemblies can be used to provide gas transferinto growth chambers. These include, for example, porous materials inthe form of tubing made of TEFLON® (PTFE), polysulfone, polypropylene,silicone, KYNAR® (PVDF), and the like. In some embodiments, used toprovide gas transfer into growth chambers. As noted above, sparger body1500 can include TYVEK® material, which can be used in a bioreactor forthe use of active gas diffusion. Similarly, this material can be used ina growth chamber utilizing passive gas transfer. Permeability of TYVEK®film can be measured using the quantitative property of Gurley HillPorosity. In some embodiments, such materials range in values betweenabout 6 to about 30 (sec/100 cc in²). Permeability rated according tothe methods of Bendtsen Air Permeability are often in a range betweenabout 400 to about 2000 (ml/min).

In some embodiments, a permeable material will have high permeabilitywhile maintaining hydrophobicity, strength, weldability,biocompatibility, and gamma stability. Often, it is desirable to have aflexible material that welds readily to common materials used in thefilm or port configurations, often found in the manufacture ofbioprocessing containers (BPCs). For example, the flexible nature of asoft or paper like film can allow it to be folded during manufacturing,packaging, loading, and use of the bioreactor. It may also be desirousto allow for the surface area and shape of the sparge material to easilybe modified or changed according to weld or cut pattern. Optionally,instead of providing a sparger body to be immersed in the contents of acontainer, a permeable envelope could be used to encapsulate the liquidcontents of the bioreactor, thus providing a broad area for diffusion.

Welding the sparger body on a port or container surface can provide fora high level of surface area while providing a low-profile sparge. Insome embodiments, this can reduce turbulence near the impeller and/orreduce the possibility of cells accumulating in cracks, seams, orcrevices. Often, conventional sparge configurations rely on the use ofsparging rings that have small hole perforations that are placed belowthe impeller. Spargers can also include the use of extremely small poresizes. Such porous materials are commonly seen as sintered metal orceramic materials. Using a single use disposable material such as TYVEK®may be helpful in avoiding or reducing contamination and cleaning issuesthat may be associated with some conventional spargers, which sometimesinvolve cleaning numerous holes, pores, and crevices of such units. Forexample, small void areas in some spargers may present areas for celldebris to lodge and accumulate leading to increased occurrence ofcontamination. In some cases, this may carry over in subsequent cellruns.

One purpose of a sparge unit in a cell culture is to aid in the masstransfer of oxygen (K_(L)a), which is often necessary for therespiration of the growing cells. An advantage of a sparge approach usedin a single use bioreactor is that the tortuous pore structure of a gaspermeable membrane such as TYVEK® can allow for a beneficial effect onmass transfer of oxygen from the bulk gas introduced through thesparger. In some embodiments, it is desirable to have small bubblesintroduced into the bioreactor as they can benefit mass transfer. Masstransfer across a permeable membrane can occur independent of masstransfer resulting from a gas bubble. Relatedly, a long gas retentiontime within the fluid column and higher surface to volume ratios areoften desirable effects. It is generally accepted that the bubble sizecan be dominated by surface tension effects, inherently related to thecomponent ratio of salts, proteins, sugars, and micro and macrocomponents of the nutrient media. Experimentally calculated K_(L)avalues, visual observation, and data from bioreactor runs often indicatethat bubble size and perhaps improved mass transfer are qualities of thepresent sparge approaches. The composition and rheological properties ofthe liquid, mixing intensity, turnover rate of the fluid, bubble size,presence of cell clumping, and interfacial absorption characteristicsall influence mass transfer of gas such as oxygen to the cells. Maindriving forces of mass transfer include surface area and concentrationgradient. In many cases, a main source of resistance of oxygen masstransfer in a stirred tank bioreactor can be the liquid film surroundingthe gas bubble.

A sparging material such as TYVEK® can provide for the transfer of gasacross the membrane. Relatedly, by incorporating TYVEK® and similar gaspermeable membranes, the surface area can easily be increased. In someembodiments, the oxygen gradient between the membrane and the liquidinterface can be maintained at a high level through constantreplenishment directly through a sparge inlet. Further, a rapid mixingintensity can also benefit mass transfer as the impeller pumps mediadirectly down onto a sparger surface. The use of a membrane can allowfor mass transfer of oxygen across the bulk film surface, which can bein addition to the formation of bubbles that rise within the fluidcolumn. In many cases, small bubbles can lead to greater foaming at thetop of a bioreactor, which can have negative effects on cell viabilityand K_(L)a according to Henry's law and the solubility of gases relatedto partial pressures. This boundary layer often results in a reducedability to control dissolved oxygen levels within the bulk liquid.Typically, it is desirable to avoid or mitigate the presence of foam, asexcessive amounts can result in exhaust filter blocking and run failure.The novel sparger approaches described herein can provide the desiredmass transfer properties, often with reduced levels of foam generated ascompared to conventional systems. This may be due to greater efficacyand less gas being introduced through the sparger to maintain a targetoxygen solubility.

TYVEK® is similar in some aspects to the material GORE-TEX® in that ithas hydrophobic qualities but will still allow water vapor to passthrough. For medical grades of TYVEK® a large relative pore size can beabout 20 (micrometers) and the surface energy can be about 25 to about32 (dynes/cm). As mentioned elsewhere herein, it may be beneficial touse a check valve in a gas inlet stream near a sparger to reduceundesirable transfer of water vapor through the membrane when thesparger is submerged while not in use. Actual moisture transmissionrates may vary largely with the media used and the particularapplication. Moisture Vapor Transmission Rates (MTVR) often range fromabout 1500 to about 1640 (g/m²/24 hrs). The present invention alsocontemplates the use of these sparger approaches in the form of areplaceable retrofit kit, which may be adapted for use with conventionalbioreactors. Such kits can improve K_(L)a and replace a piece ofhardware commonly used in steam sterilized bioreactors that may bedifficult to sterilize or clean.

It is appreciated that any of a variety of permeable membranes may beused as a sparging material. In some embodiments, such membranes may becomprised of high density polyethylene fibers that are heat sealed intoa web having a thickness in a range between about 50 microns to about250 microns. The fibers typically have a diameter in a range betweenabout 2 microns to about 8 microns and can be produced by a flash spunprocess or other methods.

In other embodiments, the sparging material may include a perforatedfilm sheet, such as a sheet of low density PE film with small perforatedholes. This may be in the form of a plastic tubing, molded plastic,shaped film, or flat film. The small perforated holes can be, forexample, punched, molded, or embossed into the film. As described above,such sparging materials or constructions can be fixed to the container.In some embodiments, a sparging mechanism may include a combination of apermeable membrane and a perforated film.

FIG. 16 illustrates a cross-section view of a sparger assembly 1600according to one embodiment of the present invention. Sparger assembly1600 can include a sheet of permeable material 1605 and a spargerconduit 1610. As shown here, sheet of permeable material 1605 is annularin shape. Sparger assembly 1600 can be in fluid communication with aport of a container (now shown) via sparger conduit 1610. An inner ring1603 and an outer ring 1604 of sheet 1605 can each be anchored to theinterior surface of a container 1602, such that the sheet of permeablematerial 1605, as coupled with container 1602, defines a donut-shapedspace.

FIG. 17 illustrates a cross-section view of a sparger assembly 1700according to one embodiment of the present invention. Sparger assembly1700 can include any number of sheets of permeable material 1705, asparger tube 1730, and a sparger conduit 1710. Sparger assembly 1700 canbe in fluid communication with a port 1720 of a container 1702 via asparger conduit 1710. As shown here, sparger assembly 1700 can include asparger body 1706 that is constructed of two sheets of permeablematerial 1705 which are coupled together along their outer rings 1704.It is appreciated that sparger body 1706 can be configured in any of avariety of shapes, including spheres, cylinders, boxes, pyramids,irregular shapes, and the like, and may include any combination ofpermeable and non-permeable materials or surfaces.

FIG. 18 illustrates a cross-section view of a sparger assembly 1800according to one embodiment of the present invention. Sparger assembly1800 can include a sheet of permeable material 1805 and a spargerconduit 1810. Sparger assembly 1800 can be in fluid communication with aport 1820 of a container 1802 via sparger conduit 1810. As shown here,sheet of permeable material 1805 is circular in shape. An outer ring1804 of sheet 1805 can each be anchored to the interior surface of acontainer 1802, such that the sheet of permeable material 1805, ascoupled with container 1802, defines a dome-shaped space. Spargerassembly configurations such as those described herein can allow thesurface area and corresponding gas flow rate requirements of, forexample, the permeable material 1805 to be adjusted by utilizingdifferent size shapes such as the dome shown here. Some embodiments ofthe present invention may include a check valve inline coupled with atubing that is attached to the sparger conduit 1810, which can preventfluid backflow.

FIG. 19 illustrates a cross-section view of a sparger assembly 1900according to one embodiment of the present invention. Sparger assembly1900 can include a sheet of permeable material 1905 and a spargerconduit 1910. Sparger assembly 1900 can be in fluid communication with aport of a container (not shown) via sparger conduit 1910. As shown here,sheet of permeable material 1905 is circular in shape. An outer ring1904 of sheet 1905 can be coupled with sparger conduit 1910, such thatthe sheet of permeable material 1905, as coupled with sparger conduit1910, defines a dome-shaped space.

FIG. 20 illustrates a partial perspective view of a reactor system 2000according to one embodiment of the present invention. Reactor system2000 can include a drive motor 2095 coupled with a drive shaft 2004.Reactor system 2000 can also include a frame support 2097 coupled withdrive motor 2095. In use, drive shaft 2004 can be coupled with arotational assembly 2001 to mix or agitate the contents of a container(not shown) which is coupled with rotational assembly 2001. In someembodiments, rotational assembly 2001 is coupled with frame support 2097via a bracket (not shown). FIG. 21 illustrates a partial perspectiveview of a reactor system 2100 according to one embodiment of the presentinvention. Reactor system 2100 can include a drive motor (not shown)coupled with a drive shaft 2104. Reactor system 2100 can also include aframe support 2197 coupled with the drive motor. Drive shaft 2104 mayinclude or be in operative association with a drive shaft ear 2006 thatis configured to couple with a notch of a rotational assembly hub (notshown). Drive shaft ear 2006 is often used to transmit torque from thedrive motor to the rotational assembly hub.

FIG. 22 illustrates a partial perspective view of a reactor system 2200according to one embodiment of the present invention. Reactor system2200 can include a drive motor 2295 coupled with a drive shaft 2204. Inuse, drive shaft 2204 can be coupled with a rotational assembly 2201 tomix or agitate the contents of a container (not shown) which is coupledwith rotational assembly 2201. A clamp 2205 may also be coupled withrotational assembly 2201. In this embodiment, drive motor 2295 includesa right angle gearmotor, which can allow an operator to pass drive shaft2204 through drive motor 2295 without moving the drive motor 2295.Embodiments that include right angle gear motors, parallel shaft gearmotors, and hollow shaft motors can provide enhanced alignment and easeof connection between drive motor 2295 and rotational assembly 2201.FIG. 23 illustrates a cross-section view of a reactor system 2300according to one embodiment of the present invention. Reactor system2300 can include a drive motor 2395 coupled with a drive shaft 2304.Drive shaft 2304 may include or be coupled with a tapered element 2304 athat is configured to associate with a corresponding receiving element2395 a of motor 2395. Tapered element 2304 a can provide enhancedalignment between drive shaft 2304 and drive motor 2395.

FIG. 24 illustrates a perspective view of a reactor system 2400according to one embodiment of the present invention. Reactor system2400 can include a container housing 2411 coupled with a support shelf2413. Support shelf 2413 may be adapted for supporting sensing probes(not shown) and other elements of a reactor system. Container housing2411 can be coupled with a drive motor 2495 via a support frame 2497.FIG. 25 illustrates a perspective view of a reactor system 2500according to one embodiment of the present invention. Reactor system2500 can include a container housing 2511 coupled with a support shelf2513. Container housing 2511 can be coupled with a drive motor 2595 viaa support frame 2597.

FIG. 26 illustrates a probe assembly 2600 according to one embodiment ofthe present invention. As seen here, probe assembly 2600 is in aretracted configuration, prior to engagement with a reactor container.Probe assembly 2600 can include a dissolved oxygen and pH probe 2610 andPALL KLEENPAK® connectors 2620 for providing an aseptic connection.Probe assembly 2600 can also include a port 2630, a sleeve 2640, and acoupler 2650, and these three components can facilitate the integrationof probe 2610 into the reactor utilizing PALL connectors 2620. In someembodiment, port 2630 and female PALL connector 2720 f can be part of orintegral with the reactor container (not shown). Sleeve 2640, coupler2650, and male PALL connector 2720 m can be manufactured or provided tothe user as a separate subassembly. The user can install the desiredprobe into such a subassembly and then can sterilize the complete probeassembly. Port 2630, sleeve 2640, and coupler 2650 can facilitateintegration of probe 2610 into a bioreactor using PALL connector 2620.

FIG. 27A provides an illustration of a probe port subassembly 2702 of aprobe assembly according to one embodiment of the present invention.Probe port subassembly 2702 can include a bioprocessing container port2730 coupled with female PALL connector 2720 f. Port 2730 may be, forexample, heat welded into a container (not shown) via flange plane 2734.Port 2730 may also include a lip seal 2732 that can prevent backflow offluid or material from the container into probe assembly or beyondflange 2734 plane. In some embodiments, port 2730 and female PALLconnector 2720 f are constructed integrally with the container.

FIG. 27B illustrates a probe kit subassembly 2704 of a probe assemblyaccording to one embodiment of the present invention. Probe kitsubassembly 2704 can include a coupler 2750, a sleeve 2740, and a malePALL connector 2720 m. Probe kit subassembly 2704 may be supplied to anend user as a separate kit. Sleeve 2740 may be coupled with coupler 2750via a barb fitting (not shown) of coupler 2750. Similarly, sleeve 2740may be coupled with male PALL connector 2720 m via a barb fitting (notshown) of male PALL connector 2720 m.

FIG. 27C illustrates an autoclave subassembly 2706 of a probe assemblyaccording to one embodiment of the present invention. Autoclavesubassembly 2706 can include a probe 2710, coupler 2750, sleeve 2740,and male PALL connector 2720 m. An end user can install the desiredprobe 2710 into a probe kit subassembly 2704 as described above, andsterilize the resulting autoclave assembly 2706. After sterilization,the user can join the male PALL connector 2720 m and the female PALLconnector 2720 f, and complete the probe engagement into the fluidstream. In some embodiments, sleeve 2740 is a flexible member that cancollapse and allow probe 2710 to be displaced, and coupler 2750 canprovide an interface between sleeve 2740 and probe 2710.

FIG. 28A illustrates a probe assembly 2800 according to one embodimentof the present invention. Probe assembly 2800 includes probe 2810,coupler 2850, sleeve 2840, male PALL connector 2820 m, female PALLconnector 2820 f, and port 2830. Probe assembly 2800 is shown in a firstconnected configuration, wherein probe assembly is engaged withcontainer, but the probe is not yet introduced into the fluid stream.FIG. 28B illustrates a probe assembly according to one embodiment of thepresent invention, wherein probe assembly 2800 is in a second connectedconfiguration such that sleeve 2840 is collapsed and a distal end ofprobe 2810 is introduced into the fluid stream of the container.

C. Cultures

The stirred-tank reactor system can be designed to hold a fluidic mediumsuch as a biological fluid, a cell culture medium, a culture ofmicroorganisms, a food production, or the like. When the fluidic mediumis a cell culture the system can be operated in, for example,batch-mode, semi-batch mode, fed-batch mode, or continuous mode. A batchculture can be a large scale cell culture in which a cell inoculum iscultured to a maximum density in a tank or fermenter, and harvested andprocessed as a batch. A fed-batch culture can be a batch culture whichis supplied with either fresh nutrients (e.g., growth-limitingsubstrates) or additives (e.g., precursors to products). A continuousculture can be a suspension culture that is continuously supplied withnutrients by the inflow of fresh medium, wherein the culture volume isusually constant. Similarly, continuous fermentation can refer to aprocess in which cells or micro-organisms are maintained in culture inthe exponential growth phase by the continuous addition of fresh mediumthat is exactly balanced by the removal of cell suspension from thebioreactor. Furthermore, the stirred-tank reactor system can be used forsuspension, perfusion or microcarrier cultures. Generally, thestirred-tank reactor system can be operated as any conventionalstirred-tank reactor with any type of agitator such as a Rushton,hydrofoil, pitched blade, or marine. With reference to FIG. 1 , theagitation shaft 112 can be mounted at any angle or position relative tothe housing 111, such as upright centered, upright offset, or 15°offset. The control of the stirred-tank reactor system can be byconventional means without the need for steam-in-place (SIP) orclean-in-place (CIP) control. In fact, the system of the instantinvention is not limited to sterile bioreactor operation, but can beused in any operation in which a clean product is to be mixed using astirred tank, for example, food production or any clean-room mixingwithout the need for a clean-room.

D. The Kit

The invention encompasses a kit that includes a stirred-tank reactorsystem and instructions for use. In one embodiment, the kit includes adisposable stirred-tank reactor system. Accordingly, the kit includes atleast one disposable element such as the bag, the shaft, the impeller,or the bearing. The kit can be entirely disposable. The flexible,disposable bag may be affixed to the shaft and the bearing through atleast one seal or o-ring such that the inside of the bag remainssterile. In addition, the bag may include a pH sensor and adissolved-oxygen sensor, wherein the sensors are incorporated into thebag and are disposable with the bag. The kit may also include one ormore internal pouches that are sealed to the bag. The pouch has one endthat can be opened to the outside of the bag such that a probe can beinserted into the reactor. The probe may be a temperature probe, a pHprobe, a dissolved gas sensor, an oxygen sensor, a carbon dioxide (CO₂)sensor, a cell mass sensor, a nutrient sensor, an osmometer, and thelike. Furthermore, the system may include at least one port in the bagallowing for the connection of a device to the port, wherein the deviceincludes, but is not limited to, a tube, a filter, a sampler, a probe, aconnector, and the like. The port allows for sampling, titration, addingof chemostat reagents, sparging, and the like. The advantage of this kitis that it is optionally entirely disposable and easy-to-use byfollowing the attached instructions. This kit comes in different sizesdepending on the preferred culture volume and can be employed with anydesired reaction chamber or barrel. This kit is pre-sterilized andrequires no validation or cleaning. The kit can be used for cellculture, culture of microorganisms, culture of plant metabolites, foodproduction, chemical production, biopharmaceutical production, andothers.

In another embodiment the kit includes a housing or barrel that holdsthe disposable bag. Such a housing or barrel can be supplied with thebag or provided separately.

E. Examples

The following specific examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims.

(1) a Disposable Bioreactor

One example of a stirred-tank reactor system of the instant invention isa disposable bioreactor, or single use bioreactor (SUB). The bioreactoris similar to a 250 liter media bag with built-in agitation andattachable sensors (e.g., pH sensors, temperature sensors, dissolvedoxygen (dO₂) sensors, etc.). The reactor is operated via conventionalcontrollers. The agitator (e.g., agitation shaft and impeller) andbearing are disposable and built into the bag. The motor attaches to asupport (e.g., motor and bearing support) or bracket(s) on the 250 literbarrel that holds the bag. In size, shape, and operation, thisbioreactor appears similar to a stainless steel reactor with a sterileliner, however, the bioreactor of this invention provides a multitude ofadvantages compared to a conventional stainless steel reactor. It can beappreciated that the size and volume of such media bags can be scaledboth upward and downward, according to industry needs.

Most importantly, the need for cleaning and steam sterilization iseliminated. The bag is pre-sterilized by irradiation and, thus, readyfor use. In fact, no cleaning, sterilization, validation or testing isrequired at culture start-up or between culture runs. Consequently, thebioreactor provides a culture environment of zero cross-contaminationbetween runs. In conventional systems, the majority of costs are relatedto clean-in-progress (CIP) and steam-in-progress (SIP) as well as thedesign of a skid and control system to oversee these functions. Thesecosts are eliminated in the disposable bioreactor and multiple productsmay be cultured or manufactured simultaneously and with much greaterease.

The disposable bioreactor can be easily scaled-up by using largerculture bags and larger barrels to hold the bags. Multiple bioreactorscan be operated at the same time without any need for extensiveengineering or cleaning. The bioreactor is a true stirred tank with wellcharacterized mixing. As such, the bioreactor has the added advantagethat it can be scaled and its contents transferred to a stainless steelreactor if desired. Notably, the bioreactor combines ease of use withlow cost and flexibility and provides, thus, a new technical platformfor cell culture.

(2) Cell Culture

The disposable bioreactor of the instant invention can be used for abatch culture in which cells are inoculated into fresh media. As thecells grow, they consume the nutrients in the media and waste productsaccumulate. For a secreted product, when the culture has run its course,cells are separated from the product by a filtration or centrifugationstep. For viral-vector production, cells are infected with a virusduring the growth phase of the culture, allowing expression of thevector followed by harvest. Since there is zero cross-contamination inthe bioreactor it works well with batch cultures.

The bioreactor can also be used for perfusion cultures, wherein productand/or waste media is continuously removed and the volume removed isreplaced with fresh media. The constant addition of fresh media, whileeliminating waste products, provides the cells with the nutrients theyrequire to achieve higher cell concentrations. Unlike the constantlychanging conditions of a batch culture, the perfusion method offers themeans to achieve and maintain a culture in a state of equilibrium inwhich cell concentration and productivity may be maintained in asteady-state condition. This can be accomplished in the disposable bagas easily as in any conventional stainless steel reactor. Forviral-vector production, the perfusion process allows for an increase inthe cell concentration and, thereby the post-infection virus titer. Fora secreted product, perfusion in the bioreactor offers the user theopportunity to increase the productivity by simply increasing the sizeof the culture bag. Most importantly, there is no need forsterilization, validation, or cleaning because the system experienceszero cross-contamination during the production process.

(3) Batch Data 1

FIG. 29 provides a graph of data that was generated using a reactorsystem according to one embodiment of the present invention. Humanembryonic kidney (HEK) 293 cells in 200 liters of CDM4 culture mediumwere incubated in a 250 liter capacity reactor system. Among otherparameters shown in the graph, the viable cell density of the reactorsystem culture increased for about the first 14 days of the batch run.

(4) Batch Data 2

FIGS. 30-34 illustrate data obtained from a single use bioreactor systemfor mammalian cell culture according to one embodiment of the presentinvention. The scaleable mass transfer characteristics of the single usestirred tank bioreactor are described. Cell growth and metabolism,antibody production, and antibody characterization data from batchculture using a 250-liter prototype system are presented and compared toresults from a traditional stainless-steel bioreactor of similar scale.

Materials and Methods—Mixing Studies. Mixing time in the bioreactor wasestimated at various agitation rates by tracking the change in pH in thereactor over time in response to addition of a base solution. Thereactor was filled to working volume of 250 liters with typical cellculture media. At time zero, 500 ml of 1N NaOH was added at the top ofthe reactor, and a combined pH glass electrode was used to measure pHfrom time zero until the pH had stabilized. The pH versus time wasplotted, and the time required to reach 95% of the final pH wasestimated from the graph.

Key scale-up parameters were determined using standard calculations thathave been well established in the chemical and pharmaceutical industry.

The mixing Reynolds number, N_(Re) is the ratio of fluid kinetic andinertial forces and is used to determine the mixing regime, eitherlaminar or turbulent:N _(Re) =ND _(i) ²ρ/μ.

The energy input into the reactor, P_(o), per volume of the reactor, V,relates to the scale at which fluid mixing and mass transfer occurs andis dependent on the impeller power number, N_(p):P _(o) /V=N _(p) ρN ³ D _(i) ⁵ /V

The impeller power number depends on the design of the impeller and is afunction of number of blades, blade width, and blade pitch. N_(p) isalso a function of the clearance of the impeller from the sides andbottom of the reactor. For various impeller types, the power number iswell documented.

Tip speed of the impeller, v_(i), relates to the fluid shear stress inthe vicinity of the impeller:V _(i) =πND _(i)

In the above equations, N=impeller rotational speed, D_(i)=impellerdiameter, ρ=fluid density, and μ=fluid viscosity.

Materials and Methods—Oxygen Transfer Studies. The volumetric oxygentransfer coefficient K_(L)a, was estimated at various agitation andsparging rates by tracking the change in dissolved oxygen, dO₂,concentration over time at the appropriate condition. The reactor wasfilled to the working volume of 250 liters with typical cell culturemedia, and a dO₂ sensor was installed in the reactor. To prepare foreach experiment, nitrogen was sparged through the bioreactor until thedO₂ concentration dropped below approximately 20% saturation with air.For each experiment, the agitation rate was set, and then air wassparged at the desired rate. The dO₂ concentration was measured versustime until it reached approximately 80% saturation with air. The valueof K_(L)a can be estimated from a graph of C_(L) versus dC_(L)/dt, basedon the following mass balance equation:dC _(L) /dt=K _(L) a(C*−C _(L))

where C_(L) is the dO₂ concentration, and C* is the equilibrium valuefor C_(L).

Materials and Methods—Cell Culture Procedures. A cell culture processthat had been developed for a traditional stainless-steel reactor of300-liter working volume was used to demonstrate the performance of thesingle use bioreactor. The cell line, media, and process parameters thathad been demonstrated in the traditional reactor were repeated in thesingle use reactor.

The cells used were CHO cells expressing a humanized monoclonalantibody. Cells were thawed and maintained in T-flasks using standardmethods. Cells were then expanded from T-flasks into custom 1-literexpansion bags prior to being introduced into a traditionalstainless-steel 110-liter inocula bioreactor. Once cells reached aconcentration of 1.6×10⁶ cells/ml, 45 liters from the traditional110-liter bioreactor were used as inocula for the single use bioreactor.Thus, exponentially growing cells from a controlled bioreactor at apre-determined cell concentration were provided as inocula for thesingle use bioreactor.

A standard, commercially available, chemically defined cell culturemedium was used. At a specified point in the batch culture, acommercially available nutrient feed that is of non-animal origin but isnot chemically defined was added. Solutions of D-glucose and L-glutaminewere added daily as required during the batch culture to maintain aconcentration of D-glucose between 1 and 3 mg/liter and a concentrationof L-glutamine between 1 and 3 mMol/liter throughout the batch.

Control of the single use bioreactor was accomplished using standard,industry-accepted sensors and controllers. The temperature, pH, and dO₂feedback controllers operated using proportional, integral, anddifferential (PID) control. Temperature was measured by a platinumresistance thermometer inserted in a thermo well in the reactor, and wascontrolled at 37° C. via an electric heat jacket. The pH was measuredusing a combined pH glass electrode that was in direct contact with thebioreactor contents. The pH was controlled at a value of 7.1 viaaddition of CO₂ into the headspace or addition of IM Na₂CO₃ to theculture. The dO₂ concentration was measured using a dO₂ sensor that wasin direct contact with the bioreactor contents. The dO₂ concentrationwas controlled at 30% saturation with air via sparging of O₂ atapproximately 0.2 liters/min. Agitation was not controlled by feedbackbut was maintained at a single set point of 110 rpm and checked daily.Level in the bioreactor was measured using a weigh scale.

A sampling system was attached to the bioreactor using a sterileconnection device, and was used to withdraw 10-ml samples as requiredduring the batch culture. Samples were withdrawn at least once daily.Samples were immediately analyzed using a Nova BioProfile 200 analyzer,which provided culture pH, dO₂, dCO₂, D-glucose, and L-glutamineconcentrations. The pH probe was standardized, as required, andD-glucose and L-glutamine solutions were added based on the Novameasurements. Viable and total cell concentrations were determined foreach sample based on hemocytometer counts using trypan blue dyeexclusion. Samples were filtered through a 0.2 μm filter and stored forlater analysis using an Igen based assay for antibody titer.

Key cell culture parameters were calculated based on the samplemeasurements. Maximum viable cell concentration, cumulative cell time atharvest, final antibody concentration, and total glucose and glutamineconsumed were calculated directly from the sample data. As a batchculture, the specific growth rate of the cells, μ, was determined foronly the exponential phase of the culture. Specific growth rate wascalculated from a regression fit of viable cell concentration, X_(v),from days one through four following inoculation:dX _(v) /dt=μt

Results from a series of batch cultures using a traditionalstainless-steel bioreactor of similar scale were available forcomparison with the single use results. The ranges of values tabulatedfor the traditional bioreactor are the 95% prediction intervals for asingle fixture observation:X _(mean) ±t _(α/2,n-1) ·s√(1+(1/n))

where x_(mean)=sample mean, s=sample standard deviation, n=sample size,and t_(α/2,n-1) is the appropriate Student's t-statistic.

The single use bioreactor supernatant was harvested, clarified byfiltration and purified (protein A-based affinity purification combinedwith ion exchange chromatography) using the procedures established forthe traditional stainless bioreactor manufacturing process. Theresultant purified antibody was characterized and compared to antibodyderived from the traditional stainless steel process. Carbohydrate (CHO)profile, SDS-PAGE (reduced and non reduced), SEC-HPLC, SEC-MALS(Multi-Angle Light Scattering), BIACore Binding, RP-HPLC, CapillaryElectrophoresis Isoelectric Focusing (CEIEF) and MALDI-TOF MassSpectrometry assays were utilized to characterize the purifiedantibodies derived from the single use bioreactor. The results obtainedwere compared to those seen for antibody produced in a traditionalstainless steel bioreactor.

Results—Mixing Studies. The time required to reach 95% homogeneitydecreased with increasing agitation speed. Each experiment was repeatedtwice, and the average mixing times are shown in Table 1.

TABLE 1 Single Use Bioreactor Mixing Studies Agitation speed (rpm) 50100 200 Characteristic mixing time (sec) 90 60 45

In addition, key scale-up parameters for the single use bioreactor couldbe readily calculated. The single use bioreactor was designed usingdesign criteria for a typical stirred tank bioreactor, and the impellerwas a typical pitched-blade design, as shown in Table 2. In the absenceof baffles, vortex formation in the reactor was avoided by mounting theimpeller at an offset from center and at a 20° angle from vertical.

TABLE 2 Single Use Bioreactor Design Elements Tank height (at workingvolume) 1.5 tank diameter Impeller diameter 0.33 tank diameter Impellernumber of blades 3   Impeller blade pitch 45°  Impeller blade height 0.5impeller diameter Impeller clearance from tank bottom 1 impellerdiameter Impeller clearance from tank side 0.5 impeller diameterImpeller power number (calculated) 2.1

Using the power number from Table 2, characteristic scale-up parameterscan be readily calculated for various agitation speeds, as listed inTable 3.

TABLE 3 Single Use Bioreactor Scale-Up Parameters Agitation speed (rpm)50 100 200 Tip speed (cm/sec) 53 106 213 Power input per unit 0.00220.018 0.143 volume(hp/1000 liter) Mixing Reynolds number 34,000 69,000137,000

Results—Oxygen Transfer Studies. The volumetric oxygen transfercoefficient, K_(L)a was determined for various flowrates of air throughthe sparger and for various agitation speeds, shown in FIG. 30 . Asexpected, K_(L)a increased with increasing air flowrate and withincreasing agitation speed, with one exception. At 200 rpm, K_(L)a waslower than that at 100 rpm. This discrepancy may be due to an increasedsurface effect on K_(L)a at the higher agitation rate. (Due to theexperimental procedure, the headspace contained a mixture of nitrogenand air.) Further experiments are required to quantify the surfaceeffects.

These results are comparable, as expected, with oxygen transfercharacteristics of traditional stirred tank bioreactors of the samegeometry. A typical literature value for the equilibrium oxygenconcentration in cell culture media is 0.18 mMol/liter, and specificoxygen uptake rate for typical animal cell culture is 0.15 mMol/10⁹cells/hr. Operated in the middle of the range from the above chart(agitation=100 rpm; sparge rate=1.0 liter/min; K_(L)a≈10 hr⁻¹) thesingle use bioreactor is calculated to be capable of maintaining cellconcentrations greater than 10×10⁶ cells/ml using air as the sparge gasand greater than 50×10⁶ cells/ml using oxygen as the sparge gas.

Results—Batch Cell Culture. To demonstrate the suitability of the singleuse bioreactor for cell culture production, CHO cells producing ahumanized monoclonal antibody were grown in batch culture and comparedto historical results from the same cell line and process carried out ina traditional stainless steel bioreactor of similar scale. This processhas been repeated five times in a 300-liter Abec traditional stainlesssteel reactor that is specifically designed for cell culture. Key cellculture parameters from the two reactors are compared in Table 4.

TABLE 4 Single Use and Traditional Bioreactor Batch Results Single UseTraditional Bioreactor Bioreactor (n = 1) (n = 5)* Duration of CellCulture (hours) 285 282 ± 8  Maximum Viable Cell Concentration 7.6  7.4± 2.4 (10₆ cells/mL) Cumulative Viable Cell Time at Harvest 1214 1019 ±171 (10⁹ cell hr/L) Specific Exponential Growth Rate 0.027  0.028 ±0.010 of Cells (1/hr) Antibody Concentration at Harvest 112 100 ± 33 (%of historical) Total Glucose Consumed (mg/L) 14.2 15.7 ± 9.4 TotalGlutamine Consumed (mMol) 16.4 18.9 ± 2.4 *range is the predictioninterval for a single future observation

The single use bioreactor was an initial prototype. As a prototype beingused for the first time, adjustments to the controller PID parameterswere made several times during the batch culture. Temporary excursionsin pH, dO₂ concentration, sparger flowrate, and agitation speed occurredat times during the batch due to these adjustments. Despite theseexcursions, results from this bioreactor are equivalent to results fromthe traditional stainless steel bioreactor. Graphs of the pH, dO₂, anddCO₂ concentration from off-line samples measured by the Nova analyzerare shown in FIG. 31 .

Detailed results from the single use bioreactor are shown in thefollowing figures. The single use bioreactor was inoculated at 0.33×10⁶cells/mL and reached a maximum cell density of 7.6×10⁶ cells/mL.Viability remained above 90% during the growth portion of the batchcurve. Total and viable cell concentration and percent viability areshown in FIG. 32 .

Antibody titer over time, as a percent of final titer at harvest, isshown in FIG. 33 . As is typical for this cell line, approximately 50%of the antibody was produced in the second half of the batch as the cellconcentration was declining.

Cumulative glucose and glutamine consumption is shown in FIG. 34 .Glucose and glutamine consumption for the single use bioreactor wascomparable to historical results from the traditional stirred tankbioreactor.

A summary of the assay results is contained in Table 5. In all cases,the antibody derived from the single use bioreactor showed equivalentresults to that produced in the traditional stainless steel bioreactor.

TABLE 5 Single Use and Traditional Bioreactor Protein Assay ResultsTraditional Single Use Assay Bioreactor Bioreactor Carbohydrate (CHO)Comparable to Comparable to profile reference reference SDS-PAGEComparable to Comparable to Reduced reference reference SDS-PAGEComparable to Comparable to Non-reduced reference reference SEC-MALS~150 KD, ~150 KD, >98% monomer >98% monomer BIACore Binding Passspecification Pass specification CEIEF Pass specification Passspecification MALDI-TOF ~150 Kd Comparable to Mass Spec. referenceRP-HPLC >95% purity (Pass) >95% purity (Pass) Peptide Mapping Comparableto reference

Various modifications and variations of the present invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of theclaims.

What is claimed is:
 1. A reactor system comprising: a support housing having an interior surface bounding a chamber, the chamber having a vertically extending central longitudinal axis; a flexible bag having an interior surface bounding a compartment, the flexible bag being disposed within the chamber of the support housing; a mixing element disposed within the compartment of the flexible bag; and a drive shaft having a first end secured to the mixing element within the compartment of the flexible bag and an opposing second end disposed outside of the flexible bag, the drive shaft having a solid core along a length thereof so that no passage is formed in the drive shaft along the length, wherein the mixing element is laterally offset by a distance from the vertically extending central longitudinal axis of the support housing, wherein the mixing element comprises an impeller having a rotational axis about which the impeller rotates, the rotational axis of the impeller being offset from the vertically extending central longitudinal axis of the support housing.
 2. The reactor system as recited in claim 1, wherein the impeller has an impeller body and a plurality of pitched impeller blades radially outwardly projecting from the impeller body, wherein the mixing element is laterally offset from the vertically extending central longitudinal axis of the support housing so that the impeller body is spaced apart from the vertically extending central longitudinal axis of the support housing.
 3. The reactor system as recited in claim 2, wherein the drive shaft projects into the impeller body.
 4. The reactor system as recited in claim 1, wherein the rotational axis is angled relative to the vertically extending central longitudinal axis of the support housing by a defined angle greater than zero.
 5. The reactor system as recited in claim 1, further comprising: the drive shaft having a central longitudinal axis extending along the length thereof; and a drive motor coupled to the drive shaft such that activation of the drive motor causes rotation of the drive shaft about the central longitudinal axis of the drive shaft.
 6. The reactor system as recited in claim 5, wherein the drive motor is coupled to the drive shaft such that activation of the drive motor causes rotation of the drive shaft about the central longitudinal axis of the drive shaft without lateral displacement of the drive shaft.
 7. The reactor system as recited in claim 1, wherein the mixing element is rotatably coupled to the flexible bag.
 8. The reactor system as recited in claim 1, further comprising: an elongated tubular connector disposed within the compartment of the flexible bag, the tubular connector having a first end connected to the bag and an opposing second end disposed within the compartment; the impeller disposed within the compartment of the flexible bag, the impeller being coupled with the second end of the tubular connector; and the drive shaft removably received within the tubular connector and removably coupled with the impeller such that rotation of the drive shaft facilitates rotation of the impeller.
 9. The reactor system as recited in claim 8, further comprising a hub rotatably connected to the flexible bag and having a passageway extending therethrough, the first end of the tubular connector being secured to the hub.
 10. A reactor system comprising: a support housing having an interior surface bounding a chamber; a flexible bag having an interior surface bounding a compartment, the flexible bag being disposed within the chamber of the support housing; an impeller disposed within the compartment of the flexible bag, the impeller having a rotational axis about which the impeller rotates; and a drive shaft secured to the impeller, the drive shaft having a solid core along a length thereof so that no passage is formed in the drive shaft along the length, wherein the rotational axis of the impeller is angled relative to vertical by a defined angle greater than zero.
 11. The reactor system as recited in claim 10, wherein the chamber of the support housing has a vertically extending central longitudinal axis, the rotational axis of the impeller being angled relative to the vertically extending central longitudinal axis by the defined angle greater than zero.
 12. The reactor system as recited in claim 10, wherein the impeller is rotatably secured to the flexible bag.
 13. The reactor system as claimed in claim 10, wherein the drive shaft rotates the impeller.
 14. The reactor system as recited in claim 10, wherein the impeller comprises an impeller body and a plurality of pitched impeller blades radially outwardly projecting from the impeller body, the drive shaft projecting into the impeller body.
 15. The reactor system as recited in claim 10, wherein the drive shaft has a central longitudinal axis being disposed at the same angle as the rotational axis.
 16. The reactor system as recited in claim 15, further comprising a drive motor, the drive motor being coupled to the drive shaft such that activation of the drive motor causes rotation of the drive shaft about the central longitudinal axis of the drive shaft without lateral displacement of the drive shaft.
 17. The reactor system as recited in claim 10, further comprising: an elongated tubular connector disposed within the compartment of the flexible bag, the tubular connector having a first end and an opposing second end; a tubular hub rotatably connected to the flexible bag, the first end of the tubular connector being connected to the hub; the impeller being disposed within the compartment of the flexible bag and coupled with the second end of the tubular connector; and the drive shaft being removably received within the tubular connector and removably coupled with the impeller such that rotation of the drive shaft facilitates rotation of the impeller.
 18. The reactor system as claimed in claim 17, wherein rotation of the drive shaft facilitates rotation of the impeller, hub, and tubular connector.
 19. The reactor system as claimed in claim 17, wherein the tubular connector comprises a flexible tube.
 20. A reactor system comprising: a support housing having an interior surface bounding a chamber, the chamber having a vertically extending central longitudinal axis; a flexible bag having an interior surface bounding a compartment, the flexible bag being disposed within the chamber of the support housing; a mixing element disposed within the compartment of the flexible bag; a drive shaft having a first end secured to the mixing element within the compartment of the flexible bag and an opposing second end disposed outside of the flexible bag, the drive shaft having a solid core along a length thereof so that no passage is formed in the drive shaft along the length; the support housing comprising a floor and a sidewall upstanding from the floor, the sidewall encircling the chamber; a side opening extending laterally through the sidewall so as to communicate with the chamber; and a support shelf radially outwardly projecting from an exterior surface of the sidewall at a location between the side opening and the floor.
 21. A reactor system comprising: a support housing having an interior surface bounding a chamber, the chamber having a vertically extending central longitudinal axis; a flexible bag having an interior surface bounding a compartment, the flexible bag being disposed within the chamber of the support housing; a mixing element disposed within the compartment of the flexible bag; a drive shaft having a first end secured to the mixing element within the compartment of the flexible bag and an opposing second end disposed outside of the flexible bag, the drive shaft having a solid core along a length thereof so that no passage is formed in the drive shaft along the length, wherein the mixing element is laterally offset by a distance from the vertically extending central longitudinal axis of the support housing; the support housing having a top opening that communicates with the chamber; a support frame secured to the support housing so as to span across the top opening; and a drive motor engaging the second end of the drive shaft, the drive motor being disposed on the support frame.
 22. The reactor system as recited in claim 21, wherein the support frame comprises a first arm and a spaced apart second arm each secured to the support housing so as to span across the top opening.
 23. A reactor system comprising: a support housing having an interior surface bounding a chamber, the chamber having a vertically extending central longitudinal axis; a flexible bag having an interior surface bounding a compartment, the flexible bag being disposed within the chamber of the support housing; a mixing element disposed within the compartment of the flexible bag; a drive shaft having a first end secured to the mixing element within the compartment of the flexible bag and an opposing second end disposed outside of the flexible bag, the drive shaft having a solid core along a length thereof so that no passage is formed in the drive shaft along the length; and a hub rotatably connected to the flexible bag and having a passageway extending therethrough, the second end of the drive shaft being disposed within the passageway of the hub.
 24. The reactor system as recited in claim 1, further comprising a rotational assembly, the rotational assembly comprising: a casing securely fixed to the flexible bag; and a hub rotatably secured to the casing and having a passageway extending therethrough, the drive shaft being received within the passageway of the hub. 